Anwendung der konfokalen Laser-Scanning-Mikroskopie zur Charakterisierung der Struktur von Zähnen und von oberflächenmodifiziertem Titan für Knochenimplantate

Zusammenfassung Mittels Fluoreszenzfarbstoffen können Strukturen sichtbar gemacht werden, die auf kon-ventionellem Weg nicht, oder nur schwer darzustellen sind. Besonders in Kombination mit der Konfokalen Laser Scanning Mikroskopie eröffnen sich neue Wege zum spezifischen Nachweis unterschiedlichster Komponenten biologischer Proben und gegebenenfalls deren dreidimensionale Widergabe.Die Visualisierung des Proteinanteils des Zahnhartgewebes kann mit Hilfe chemisch kopplungsfähiger Fluorochrome durchgeführt werden. Um zu zeigen, daß es sich bei dieser Markierung nicht um unspezifische Adsorption des Farbstoffes handelt, wurde zur Kontrolle die Proteinkomponente der Zahnproben durch enzymatischen Verdau beseitigt. Derartig behandelte Präparate wiesen eine sehr geringe Anfärbbarkeit auf.Weiterführend diente diese enzymatische Methode als Negativkontrolle zum Nachweis der Odontoblastenfortsätze im Dentin bzw. im Bereich der Schmelz-Dentin-Grenze. Hiermit konnte differenziert werden zwischen reinen Reflexionsbildern der Dentinkanäle und den Zellausläufern deren Membranen gezielt durch lipophile Fluoreszenzfarbstoffe markiert wurden.In einem weiteren Ansatz konnte gezeigt werden, daß reduzierte und daher nichtfluoreszente Fluoresceinabkömmlinge geeignet sind, die Penetration von Oxidationsmitteln (hier H2O2) in den Zahn nachzuweisen. Durch Oxidation dieser Verbindungen werden fluoreszierende Produkte generiert, die den Nachweis lieferten, daß die als Zahnbleichmittel eingesetzten Mittel rasch durch Schmelz und Dentin bis in die Pulpahöhle gelangen können.Die Abhängigkeit der Fluoreszenz bestimmter Fluorochrome von deren chemischer Um-gebung, im vorliegenden Fall dem pH-Wert, sollte eingesetzt werden, um den Säuregrad im Zahninneren fluoreszenzmikroskopisch darzustellen. Hierbei wurde versucht, ein ratio-metrisches Verfahren zu entwickeln, mit dem die pH-Bestimmung unter Verwendung eines pH-abhängigen und eines pH-unabhängigen Fluorochroms erfolgt. Diese Methode konnte nicht für diese spezielle Anwendung verifiziert werden, da Neutralisationseffekte der mineralischen Zahnsubstanz (Hydroxylapatit) die pH-Verteilung innerhalb der Probe beeinflußen. Fluoreszenztechniken wurden ebenfalls ergänzend eingesetzt zur Charakterisierung von kovalent modifizierten Implantatoberflächen. Die, durch Silanisierung von Titantestkörpern mit Triethoxyaminopropylsilan eingeführten freien Aminogruppen konnten qualitativ durch den Einsatz eines aminspezifischen Farbstoffes identifiziert werden. Diese Art der Funktionalisierung dient dem Zweck, Implantatoberflächen durch chemische Kopplung adhäsionsvermittelnder Proteine bzw. Peptide dem Einheilungsprozeß von Implantaten in den Knochen zugänglicher zu machen, indem knochenbildende Zellen zu verbessertem Anwachsverhalten stimuliert werden. Die Zellzahlbestimmung im Adhäsionstest wurde ebenfalls mittels Fluoreszenzfarbstoffen durchgeführt und lieferte Ergebnisse, die belegen, daß die durchgeführte Modifizierung einen günstigen Einfluß auf die Zelladhäsion besitzt.

Konformationsanalyse und Lipidbindung am rekombinanten Lichtsammlerprotein LHCIIb höherer Pflanzen

Die Struktur des Haupt-Lichtsammlerproteins II (LHCIIb) höherer Pflanzen ist aufgrund kristallographischer Strukturanalysen zu 94% aufgeklärt. Dennoch ist es bislang nicht gelungen, die aminoterminale Region des Komplexes vollständig zu lokalisieren. In einem ersten Abschnitt dieser Dissertation sollte anhand einer vergleichenden Bindungsstudie mit Hilfe von in vitro - Rekonstitutionen des LHCIIb geklärt werden, ob es sich bei dem so genannten N - terminalen Trimerisierungsmotiv des Lichtsammlerproteins um eine Interaktionsstelle mit dem Phospholipid Phosphatidylglyzerin handelt. Dazu wurden mehrere vergleichende Lipidbindungsstudien an rekombinantem Wildtyp - Protein und verschiedenen LHCIIb - Trimerisierungsmotiv - Mutanten durchgeführt, die allerdings nicht zu reproduzierbaren Ergebnissen führten.Im zweiten Teil dieser Arbeit wurden intra- und intermolekulare Distanzmessungen an rekombinantem LHCIIb mit Hilfe der Elektronenspin - Resonanz - Spektroskopie durchgeführt. Dazu wurden zweifach mit einem Spin - Label markierte LHCIIb - Monomere und Trimere mit je einer Markierungsposition pro Monomer benutzt. Im Anschluss an die Messungen wurden die erhaltenen Distanzinformationen zusammen mit den bereits zugänglichen Kristallstrukturdaten des Komplexes für eine Modellierung der aminoterminalen Region des LHCIIb verwendet. Die resultierenden Modelle lassen den Schluss zu, dass es im LHCIIb - Trimer zu konformativen Restriktionen des Aminoterminus kommt. Dem entgegen findet man eine größere konformative Diversität in den vermessenen monomeren Komplexen.

Photoreactive polyorganosiloxane nanoparticles and the fabrication of photocleavable microcapsules

In this thesis, we have presented the preparation of highly crosslinked spherical photoreactive colloidal particles of radius about 10 nm based on the monomer trimethoxysilane. These particles are labeled chemically with two different dye systems (coumarin, cinnamate) which are known to show reversible photodimerization. By analyzing the change in particle size upon UV irradiation with dynamic light scattering, we could demonstrate that the partially reversible photoreaction in principle can be utilized to control increase and decrease of colloidal clusters. Here, selection of the appropriate wavelengths during the irradiation employing suitable optical filters proved to be very important. Next, we showed how photocrosslinking of our nanoparticles within the micrometer-sized thin oil shell of water-oil-water emulsion droplets leads to a new species of optically addressable microcontainers. The inner water droplet of these emulsions may contain drugs, dyes or other water-soluble components, leading to filled containers. Thickness, mechanical stability and light resistance of the container walls can be controlled in a simple way by the amount and adjustable photoreactivity (= No. of labels/particle) of the nanoparticles. Importantly, the chemical bonds between the nanoparticles constituting the microcapsule shell can be cleaved photochemically by irradiation with uv light. An additional major advantage is that filling our microcapsules with water-soluble substrate molecules is extremely simple using a solution of the guest molecules as inner water phase of the W/O/W-emulsion. This optically controlled destruction of our microcontainers thus opens up a pathway to controlled release of the enclosed components as illustrated by the example of enclosed cyclodextrin molecules.

Supramolecular approaches to organized luminescent nanostructures for sensing, labeling and imaging applications

The common thread of this thesis is the will of investigating properties and behavior of assemblies. Groups of objects display peculiar properties, which can be very far from the simple sum of respective components’ properties. This is truer, the smaller is inter-objects distance, i.e. the higher is their density, and the smaller is the container size. “Confinement” is in fact a key concept in many topics explored and here reported. It can be conceived as a spatial limitation, that yet gives origin to unexpected processes and phenomena based on inter-objects communication. Such phenomena eventually result in “non-linear properties”, responsible for the low predictability of large assemblies. Chapter 1 provides two insights on surface chemistry, namely (i) on a supramolecular assembly based on orthogonal forces, and (ii) on selective and sensitive fluorescent sensing in thin polymeric film. In chapters 2 to 4 confinement of molecules plays a major role. Most of the work focuses on FRET within core-shell nanoparticles, investigated both through a simulation model and through experiments. Exciting results of great applicative interest are drawn, such as a method of tuning emission wavelength at constant excitation, and a way of overcoming self-quenching processes by setting up a competitive deactivation channel. We envisage applications of these materials as labels for multiplexing analysis, and in all fields of fluorescence imaging, where brightness coupled with biocompatibility and water solubility is required. Adducts of nanoparticles and molecular photoswitches are investigated in the context of superresolution techniques for fluorescence microscopy. In chapter 5 a method is proposed to prepare a library of functionalized Pluronic F127, which gives access to a twofold “smart” nanomaterial, namely both (i)luminescent and (ii)surface-functionalized SCSSNPs. Focus shifts in chapter 6 to confinement effects in an upper size scale. Moving from nanometers to micrometers, we investigate the interplay between microparticles flowing in microchannels where a constriction affects at very long ranges structure and dynamics of the colloidal paste.

Synthetic strategies of new molecular paramagnetic mechanically-interlocked complexes

Supramolecular chemistry is a multidisciplinary field which impinges on other disciplines, focusing on the systems made up of a discrete number of assembled molecular subunits. The forces responsible for the spatial organization are intermolecular reversible interactions. The supramolecular architectures I was interested in are Rotaxanes, mechanically-interlocked architectures consisting of a "dumbbell shaped molecule", threaded through a "macrocycle" where the stoppers at the end of the dumbbell prevent disassociation of components and catenanes, two or more interlocked macrocycles which cannot be separated without breaking the covalent bonds. The aim is to introduce one or more paramagnetic units to use the ESR spectroscopy to investigate complexation properties of these systems cause this technique works in the same time scale of supramolecular assemblies. Chapter 1 underlines the main concepts upon which supramolecular chemistry is based, clarifying the nature of supramolecular interactions and the principles of host-guest chemistry. In chapter 2 it is pointed out the use of ESR spectroscopy to investigate the properties of organic non-covalent assemblies in liquid solution by spin labels and spin probes. The chapter 3 deals with the synthesis of a new class of p-electron-deficient tetracationic cyclophane ring, carrying one or two paramagnetic side-arms based on 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) moiety. In the chapter 4, the Huisgen 1,3-dipolar cycloaddition is exploited to synthesize rotaxanes having paramagnetic cyclodextrins as wheels. In the chapter 5, the catalysis of Huisgen’s cycloaddition by CB[6] is exploited to synthesize paramagnetic CB[6]-based [3]-rotaxanes. In the chapter 6 I reported the first preliminary studies of Actinoid series as a new class of templates in catenanes’ synthesis. Being f-block elements, so having the property of expanding the valence state, they constitute promising candidates as chemical templates offering the possibility to create a complex with coordination number beyond 6.

Synthesis and surface modification of semiconductor nanocrystals

The last decade has witnessed an exponential growth of activities in the field of nanoscience and nanotechnology worldwide, driven both by the excitement of understanding new science and by the potential hope for applications and economic impacts. The largest activity in this field up to date has been in the synthesis and characterization of new materials consisting of particles with dimensions in the order of a few nanometers, so-called nanocrystalline materials. [1-8] Semiconductor nanomaterials such as III/V or II/VI compound semiconductors exhibit strong quantum confinement behavior in the size range from 1 to 10 nm. Therefore, preparation of high quality semiconductor nanocrystals has been a challenge for synthetic chemists, leading to the recent rapid progress in delivering a wide variety of semiconducting nanomaterials. Semiconductor nanocrystals, also called quantum dots, possess physical properties distinctly different from those of the bulk material. Typically, in the size range from 1 to 10 nm, when the particle size is changed, the band gap between the valence and the conduction band will change, too. In a simple approximation a particle in a box model has been used to describe the phenomenon[9]: at nanoscale dimensions the degenerate energy states of a semiconductor separate into discrete states and the system behaves like one big molecule. The size-dependent transformation of the energy levels of the particles is called “quantum size-effect”. Quantum confinement of both the electron and hole in all three dimensions leads to an increase in the effective bandgap of the material with decreasing crystallite size. Consequently, both the optical absorption and emission of semiconductor nanaocrystals shift to the blue (higher energies) as the size of the particles gets smaller. This color tuning is well documented for CdSe nanocrystals whose absorption and emission covers almost the whole visible spectral range. As particle sizes become smaller the ratio of surface atoms to those in the interior increases, which has a strong impact on particle properties, too. Prominent examples are the low melting point [8] and size/shape dependent pressure resistance [10] of semiconductor nanocrystals. Given the size dependence of particle properties, chemists and material scientists now have the unique opportunity to change the electronic and chemical properties of a material by simply controlling the particle size. In particular, CdSe nanocrystals have been widely investigated. Mainly due to their size-dependent optoelectronic properties [11, 12] and flexible chemical processibility [13], they have played a distinguished role for a number of seminal studies [11, 12, 14, 15]. Potential technical applications have been discussed, too. [8, 16-27] Improvement of the optoelectronic properties of semiconductor nanocrystals is still a prominent research topic. One of the most important approaches is fabricating composite type-I core-shell structures which exhibit improved properties, making them attractive from both a fundamental and a practical point of view. Overcoating of nanocrystallites with higher band gap inorganic materials has been shown to increase the photoluminescence quantum yields by eliminating surface nonradiative recombination sites. [28] Particles passivated with inorganic shells are more robust than nanocrystals covered by organic ligands only and have greater tolerance to processing conditions necessary for incorporation into solid state structures or for other applications. Some examples of core-shell nanocrystals reported earlier include CdS on CdSe [29], CdSe on CdS, [30], ZnS on CdS, [31] ZnS on CdSe[28, 32], ZnSe on CdSe [33] and CdS/HgS/CdS [34]. The characterization and preparation of a new core-shell structure, CdSe nanocrystals overcoated by different shells (CdS, ZnS), is presented in chapter 4. Type-I core-shell structures as mentioned above greatly improve the photoluminescence quantum yield and chemical and photochemical stability of nanocrystals. The emission wavelengths of type-I core/shell nanocrystals typically only shows a small red-shift when compared to the plain core nanocrystals. [30, 31, 35] In contrast to type-I core-shell nanocrystals, only few studies have been conducted on colloidal type-II core/shell structures [36-38] which are characterized by a staggered alignment of conduction and valence bands giving rise to a broad tunability of absorption and emission wavelengths, as was shown for CdTe/CdSe core-shell nanocrystals. [36] The emission of type-II core/shell nanocrystals mainly originates from the radiative recombination of electron-hole pairs across the core-shell interface leading to a long photoluminescence lifetime. Type-II core/shell nanocrystals are promising with respect to photoconduction or photovoltaic applications as has been discussed in the literature.[39] Novel type-II core-shell structures with ZnTe cores are reported in chapter 5. The recent progress in the shape control of semiconductor nanocrystals opens new fields of applications. For instance, rod shaped CdSe nanocrystals can enhance the photo-electro conversion efficiency of photovoltaic cells, [40, 41] and also allow for polarized emission in light emitting diodes. [42, 43] Shape control of anisotropic nanocrystals can be achieved by the use of surfactants, [44, 45] regular or inverse micelles as regulating agents, [46, 47] electrochemical processes, [48] template-assisted [49, 50] and solution-liquid-solution (SLS) growth mechnism. [51-53] Recently, formation of various CdSe nanocrystal shapes has been reported by the groups of Alivisatos [54] and Peng, [55] respectively. Furthermore, it has been reported by the group of Prasad [56] that noble metal nanoparticles can induce anisotropic growth of CdSe nanocrystals at lower temperatures than typically used in other methods for preparing anisotropic CdSe structures. Although several approaches for anisotropic crystal growth have been reported by now, developing new synthetic methods for the shape control of colloidal semiconductor nanocrystals remains an important goal. Accordingly, we have attempted to utilize a crystal phase control approach for the controllable synthesis of colloidal ZnE/CdSe (E = S, Se, Te) heterostructures in a variety of morphologies. The complex heterostructures obtained are presented in chapter 6. The unique optical properties of nanocrystals make them appealing as in vivo and in vitro fluorophores in a variety of biological and chemical investigations, in which traditional fluorescence labels based on organic molecules fall short of providing long-term stability and simultaneous detection of multiple emission colours [References]. The ability to prepare water soluble nanocrystals with high stability and quantum yield has led to promising applications in cellular labeling, [57, 58] deep-tissue imaging, [59, 60] and assay labeling [61, 62]. Furthermore, appropriately solubilized nanocrystals have been used as donors in fluorescence resonance energy transfer (FRET) couples. [63-65] Despite recent progress, much work still needs to be done to achieve reproducible and robust surface functionalization and develop flexible (bio-) conjugation techniques. Based on multi-shell CdSe nanocrystals, several new solubilization and ligand exchange protocols have been developed which are presented in chapter 7. The organization of this thesis is as follows: A short overview describing synthesis and properties of CdSe nanocrystals is given in chapter 2. Chapter 3 is the experimental part providing some background information about the optical and analytical methods used in this thesis. The following chapters report the results of this work: synthesis and characterization of type-I multi-shell and type-II core/shell nanocrystals are described in chapter 4 and chapter 5, respectively. In chapter 6, a high–yield synthesis of various CdSe architectures by crystal phase control is reported. Experiments about surface modification of nanocrystals are described in chapter 7. At last, a short summary of the results is given in chapter 8.

EPR spectroscopic investigation of membrane protein structure and folding on light harvesting complex LHCIIb

Structure and folding of membrane proteins are important issues in molecular and cell biology. In this work new approaches are developed to characterize the structure of folded, unfolded and partially folded membrane proteins. These approaches combine site-directed spin labeling and pulse EPR techniques. The major plant light harvesting complex LHCIIb was used as a model system. Measurements of longitudinal and transversal relaxation times of electron spins and of hyperfine couplings to neighboring nuclei by electron spin echo envelope modulation(ESEEM) provide complementary information about the local environment of a single spin label. By double electron electron resonance (DEER) distances in the nanometer range between two spin labels can be determined. The results are analyzed in terms of relative water accessibilities of different sites in LHCIIb and its geometry. They reveal conformational changes as a function of micelle composition. This arsenal of methods is used to study protein folding during the LHCIIb self assembly and a spatially and temporally resolved folding model is proposed. The approaches developed here are potentially applicable for studying structure and folding of any protein or other self-assembling structure if site-directed spin labeling is feasible and the time scale of folding is accessible to freeze-quench techniques.

Untersuchung der Struktur und Assemblierung des Lichtsammelkomplexes II höherer Pflanzen mittels elektronenparamagnetischer Resonanz (EPR)

Der Haupt-Lichtsammelkomplex (LHCII) des Photosyntheseapparates höherer Pflanzen gehört zu den häufigsten Membranproteinen der Erde. Seine Kristallstruktur ist bekannt. Das Apoprotein kann rekombinant in Escherichia coli überexprimiert und somit molekularbiologisch vielfältig verändert werden. In Detergenzlösung besitzt das denaturierte Protein die erstaunliche Fähigkeit, sich spontan zu funktionalen Protein-Pigment-Komplexen zu organisieren, welche strukturell nahezu identisch sind mit nativem LHCII. Der Faltungsprozess findet in vitro im Zeitbereich von Sekunden bis Minuten statt und ist abhängig von der Bindung der Cofaktoren Chlorophyll a und b sowie verschiedenen Carotinoiden.rn Diese Eigenschaften machen LHCII besonders geeignet für Strukturuntersuchungen mittels der elektronenparamagnetischen Resonanz (EPR)-Spektrokopie. Diese setzt eine punktspezifische Spinmarkierung des LHCII voraus, die in dieser Arbeit zunächst optimiert wurde. Einschließlich der Beiträge Anderer stand eine breite Auswahl von über 40 spinmarkierten Mutanten des LHCII bereit, einen N-terminalen „Cys walk“ eingeschlossen. Weder der hierfür notwendige Austausch einzelner Aminosäuren noch die Anknüpfung des Spinmarkers beeinträchtigten die Funktion des LHCII. Zudem konnte ein Protokoll zur Präparation heterogen spinmarkierter LHCII-Trimere entwickelt werden, also von Trimeren, die jeweils nur ein Monomer mit einer Spinmarkierung enthalten.rn Spinmarkierte Proben des Detergenz-solubilisierten LHCII wurden unter Verwendung verschiedener EPR-Techniken strukturell analysiert. Als besonders aussagekräftig erwies sich die Messung der Wasserzugänglichkeit einzelner Aminosäurepositionen anhand der Electron Spin Echo Envelope Modulation (ESEEM). In Kombination mit der etablierten Double Electron-Electron Resonance (DEER)-Technik zur Detektion von Abständen zwischen zwei Spinmarkern wurde der membranständige Kernbereich des LHCII in Lösung eingehend untersucht und strukturell der Kristallstruktur für sehr ähnlich befunden. Die Vermessung kristallographisch nicht erfasster Bereiche nahe dem N-Terminus offenbarte die schon früher detektierte Strukturdynamik der Domäne in Abhängigkeit des Oligomerisierungsgrades. Der neue, noch zu vervollständigende Datensatz aus Abstandsverteilungen und ESEEM-Wasserzugänglichkeiten monomerer wie trimerer Proben sollte in naher Zukunft die sehr genaue Modellierung der N-terminalen Domäne des LHCII ermöglichen.rn In einem weiteren Abschnitt der Arbeit wurde die Faltung des LHCII-Apoproteins bei der LHCII-Assemblierung in vitro untersucht. Vorausgegangene fluoreszenzspektroskopi-sche Arbeiten hatten gezeigt, dass die Bindung von Chlorophyll a und b in aufeinanderfolgenden Schritten im Zeitbereich von weniger als einer Minute bzw. mehreren Minuten erfolgten. Sowohl die Wasserzugänglichkeit einzelner Aminosäurepositionen als auch Spin-Spin-Abstände änderten sich in ähnlichen Zeitbereichen. Die Daten deuten darauf hin, dass die Ausbildung der mittleren Transmembran-Helix mit der schnelleren Chlorophyll-a-Bindung einhergeht, während sich die Superhelix aus den beiden anderen Transmembranhelices erst im langsameren Schritt, zusammen mit der Chlorophyll-b-Bindung, ausbildet.rn

Institutional Processes and Discursive Strategies: Rhetoric and Vocabulary Analysis of CSR and Sustainability

The candidate tackled an important issue in contemporary management: the role of CSR and Sustainability. The research proposal focused on a longitudinal and inductive research, directed to specify the evolution of CSR and contribute to the new institutional theory, in particular institutional work framework, and to the relation between institutions and discourse analysis. The documental analysis covers all the evolution of CSR, focusing also on a number of important networks and associations. Some of the methodologies employed in the thesis have been employed as a consequence of data analysis, in a truly inductive research process. The thesis is composed by two section. The first section mainly describes the research process and the analyses results. The candidates employed several research methods: a longitudinal content analysis of documents, a vocabulary research with statistical metrics as cluster analysis and factor analysis, a rhetorical analysis of justifications. The second section puts in relation the analysis results with theoretical frameworks and contributions. The candidate confronted with several frameworks: Actor-Network-Theory, Institutional work and Boundary Work, Institutional Logic. Chapters are focused on different issues: a historical reconstruction of CSR; a reflection about symbolic adoption of recurrent labels; two case studies of Italian networks, in order to confront institutional and boundary works; a theoretical model of institutional change based on contradiction and institutional complexity; the application of the model to CSR and Sustainability, proposing Sustainability as a possible institutional logic.

New biosensor applications of surface plasmon and hydrogel optical waveguide spectroscopy

Rapid and sensitive detection of chemical and biological analytes becomes increasingly important in areas such as medical diagnostics, food control and environmental monitoring. Optical biosensors based on surface plasmon resonance (SPR) and optical waveguide spectroscopy have been extensively pushed forward in these fields. In this study, we combine SPR, surface plasmon-enhanced fluorescence spectroscopy (SPFS) and optical waveguide spectroscopy with hydrogel thin film for highly sensitive detection of molecular analytes.rnrnA novel biosensor based on SPFS which was advanced through the excitation of long range surface plasmons (LRSPs) is reported in this study. LRSPs are special surface plasmon waves propagating along thin metal films with orders of magnitude higher electromagnetic field intensity and lower damping than conventional SPs. Therefore, their excitation on the sensor surface provides further increased fluorescence signal. An inhibition immunoassay based on LRSP-enhanced fluorescence spectroscopy (LRSP-FS) was developed for the detection of aflatoxin M1 (AFM1) in milk. The biosensor allowed for the detection of AFM1 in milk at concentrations as low as 0.6 pg mL-1, which is about two orders of magnitude lower than the maximum AFM1 residue level in milk stipulated by the European Commission legislation.rnrnIn addition, LRSPs probe the medium adjacent to the metallic surface with more extended evanescent field than regular SPs. Therefore, three-dimensional binding matrices with up to micrometer thickness have been proposed for the immobilization of biomolecular recognition elements with large surface density that allows to exploit the whole evanescent field of LRSP. A photocrosslinkable carboxymethyl dextran (PCDM) hydrogel thin film is used as a binding matrix, and it is applied for the detection of free prostate specific antigen (f-PSA) based on the LRSP-FS and sandwich immunoassay. We show that this approach allows for the detection of f-PSA at low femto-molar range, which is approximately four orders of magnitude lower than that for direct detection of f-PSA based on the monitoring of binding-induced refractive index changes.rnrnHowever, a three dimensional hydrogel binding matrix with micrometer thickness can also serve as an optical waveguide. Based on the measurement of binding-induced refractive index changes, a hydrogel optical waveguide spectroscopy (HOWS) is reported for a label-free biosensor. This biosensor is implemented by using a SPR optical setup in which a carboxylated poly(N-isoproprylacrylamide) (PNIPAAm) hydrogel film is attached on a metallic surface and modified by protein catcher molecules. Compared to regular SPR biosensor with thiol self-assembled monolayer (SAM), HOWS provides an order of magnitude improved resolution in the refractive index measurements and enlarged binding capacity owing to its low damping and large swelling ratio, respectively. A model immunoassay experiment revealed that HOWS allowed detection of IgG molecules with a 10 pM limit of detection (LOD) that was five-fold lower than that achieved for SPR with thiol SAM. For the high capacity hydrogel matrix, the affinity binding was mass transport limited.rnrnThe mass transport of target molecules to the sensor surface can play as critical a role as the chemical reaction itself. In order to overcome the diffusion-limited mass transfer, magnetic iron oxide nanoparticles were employed. The magnetic nanoparticles (MNPs) can serve both as labels providing enhancement of the refractive index changes, and “vehicles” for rapidly delivering the analytes from sample solution to an SPR sensor surface with a gradient magnetic field. A model sandwich assay for the detection of β human chorionic gonadotropin (βhCG) has been utilized on a gold sensor surface with metallic diffraction grating structure supporting the excitation of SPs. Various detection formats including a) direct detection, b) sandwich assay, c) MNPs immunoassay without and d) with applied magnetic field were compared. The results show that the highly-sensitive MNPs immunoassay improves the LOD on the detection of βhCG by a factor of 5 orders of magnitude with respect to the direct detection.rn

Design und Synthese maßgeschneiderter funktioneller Rylenfarbstoffe

Die vorgelegte Dissertation beschäftigt sich mit der Darstellung und Untersuchung von funktionellen Farbstoffen auf der Basis von Rylendiimiden. Diese Substanzklasse zeichnet sich durch gute Funktionalisierbarkeit, hohe chemische und photochemische Stabilität sowie durch hohe Fluoreszenzquantenausbeuten und Extinktionskoeffizienten aus. Rylendiimide spielen eine bedeutsame Rolle in den modernen Materialwissenschaften. Ein Schwerpunkt der Arbeit liegt auf der Synthese und Untersuchung von neuen Perylendiimiden (PDI) im Hinblick auf eine Anwendung in der organischen Elektronik. Das Substitutionsmuster der PDI hat einen signifikanten Einflussrnauf deren supramolekulares Verhalten und die Leistungsfähigkeit in elektronischen Bauteilen. Durch das Einführen neuer Substituenten konnten weitergehende Erkenntnisse über das supramolekulare Verhalten der PDI gewonnenrnwerden. Multichromophore sind wichtige Modellsysteme zur Untersuchung vonrnEnergietransportprozessen und Einzelphotonenquellen. Daher liegt ein weiterer Schwerpunkt auf der Synthese multichromophorer PDI-Systeme. Neben der Darstellung definierter dendritischer Nanoteilchen auf Basis von Poly(phenylenethinylen)-Dendrimeren beschäftigt sich dieser Teil auch mit der Synthese hochverzweigter ethinylverknüpfter Polymere auf Basis von PDI-Monomeren. Aufgrund ihrer außergewöhnlichen photochemischen Eigenschaften spielen Perylen- und Terrylendiimide eine wichtige Rolle als Fluoreszenzmarker.rnDaher beschäftigt sich ein weiterer Aspekt dieser Arbeit mit der Synthese vonrnFluoreszenzmarkern, die sowohl in Polymerisationsreaktionen als auch inrnbiologischen Systemen Anwendung finden können.

Fresh Produce Safety and Good Agricultural Practices: Stakeholders' Perception and Consumers' Choice in Thailand

Concerns of Thai consumers on food safety have been recently increasing, especially in urban areas and for fresh produce because food safety scandals, such as chemical residues on fresh produce (e.g., cabbage) still frequently occur. The Thai government tried to meet consumer needs by imposing in the domestic market a stronger regulation aimed at increasing the baseline level of food safety assurance and by introducing a voluntary standard (based on Good Agricultural Practices or GAPs and known as Q-GAP) and the related food safety label (i.e., Q mark). However, since standards and regulations are weakly implemented in the domestic market compared to exported products, there is still a lack of Thai consumers’ confidence in the safety of local food products. In this work the current situation of GAPs adoption in Thai fresh produce production is analysed. Furthermore, it is studied whether Thai consumers place value on food safety labels available on the market, to know whether consumer demand could drive the market of certified safer products. This study contains three essays: 1) a review of the literature, 2) a qualitative study on stakeholders' perception toward GAPs adoption and 3) a quantitative study, aimed at analysing consumers' preferences and willingness-to-pay for food safety labels on fresh produce using a discrete choice experiment. This dissertation contributes to the economics of quality assurance and labelling, specifically addressing GAPs and food safety label in the fresh produce supply chain. Results show that Q-GAP could be effectively used to improve food safety in Thai domestic market, but its credibility should be improved. Stakeholder’s awareness toward food safety issues and the delivery of reliable and sound information are crucial. Thai consumers are willing to pay a premium price for food safety labelled produce over unlabelled ones. Implications for both government and business decision-makers are discussed.

Fluctuation spectroscopy of single plasmonic nanostructures

Plasmonic nanoparticles are great candidates for sensing applications with optical read-out. Plasmon sensing is based on the interaction of the nanoparticle with electromagnetic waves where the particle scatters light at its resonance wavelength. This wavelength depends on several intrinsic factors like material, shape and size of the nanoparticle as well as extrinsic factors like the refractive index of the surrounding medium. The latter allows the nanoparticle to be used as a sensor; changes in the proximate environment can be directly monitored by the wavelength of the emitted light. Due to their minuscule size and high sensitivity this allows individual nanoparticles to report on changes in particle coverage.rnrnTo use this single particle plasmon sensor for future sensing applications it has to meet the demand for detection of incidents on the single molecule level, such as single molecule sensing or even the detection of conformational changes of a single molecule. Therefore, time resolution and sensitivity have to be enhanced as today’s measurement methods for signal read-out are too slow and not sensitive enough to resolve these processes. This thesis presents a new experimental setup, the 'Plasmon Fluctuation Setup', that leads to tremendous improvements in time resolution and sensitivity. This is achieved by implementation of a stronger light source and a more sensitive detector. The new setup has a time resolution in the microsecond regime, an advancement of 4-6 orders of magnitude to previous setups. Its resonance wavelength stability of 0.03 nm, measured with an exposure time of 10 ms, is an improvement of a factor of 20 even though the exposure time is 3000 times shorter than in previous reports. Thus, previously unresolvable wavelength changes of the plasmon sensor induced by minor local environmental alteration can be monitored with extremely high temporal resolution.rnrnUsing the 'Plasmon Fluctuation Setup', I can resolve adsorption events of single unlabeled proteins on an individual nanorod. Additionally, I monitored the dynamic evolution of a single protein binding event on a millisecond time scale. This feasibility is of high interest as the role of certain domains in the protein can be probed by a study of modified analytes without the need for labels possibly introducing conformational or characteristic changes to the target. The technique also resolves equilibrium fluctuations in the coverage, opening a window into observing Brownian dynamics of unlabeled macromolecules. rnrnA further topic addressed in this thesis is the usability of the nanoruler, two nanospheres connected with a spacer molecule, as a stiffness sensor for the interparticle linker under strong illumination. Here, I discover a light induced collapse of the nanoruler. Furthermore, I exploit the sensing volume of a fixed nanorod to study unlabeled analytes diffusing around the nanorod at concentrations that are too high for fluorescence correlation spectroscopy but realistic for biological systems. Additionally, local pH sensing with nanoparticles is achieved.

Photo-induced reversible assembly of colloidal polymer nanoparticles

Polymer nanoparticles functionalized on the surface with photo-responsive labels were synthesized. In a first synthetic step, polystyrene was copolymerized with the cross-linker divinylbenzene and poly(ethylene glycol) acrylate in a miniemulsion, to produce nano-sized spheres (~ 60 nm radius) with terminal hydroxyl groups, which were functionalized in a subsequent synthetic step with photo-responsive labels. For this purpose, two photo-active molecular structures were separately used: anthracene, which is well known to form covalently bonded dimers upon photo-excitation; and pyrene, which only forms short lived excited state dimers (excimers). Acid derivatives of these labels (9-anthracene carboxylic acid and 1-pyrene butyric acid) were bonded to the hydroxyl terminal groups of the nanoparticles through an esterification reaction, via the intermediate formation of the corresponding acid chloride.rnThe obtained labeled nanoparticles appeared to be highly hydrophobic structures. They formed lyophobic suspensions in water, which after analysis by dynamic light scattering (DLS) and ultramicroscopic particle tracking, appeared to equilibrate as a collection of singly dispersed nanoparticles, together with a few nanoparticle aggregates. The relative amount of aggregates decreased with increasing amounts of the surfactant sodium dodecyl sulfate (SDS), thus confirming that aggregation is an equilibrated state resulting from lyophobicity. The formation of such aggregates was corroborated using scanning electron microscopy (SEM). The photo-irradiation of the lyophobic aqueous suspensions of anthracene labeled nanoparticles (An-NP) resulted in the formation of higher aggregates, as evidenced by DLS and ultramicroscopy. The obtained state of aggregation could be reverted by sonication. The possibility to re-aggregate the system in subsequent photo-excitation and sonication cycles was established. Likewise, the photo-irradiation of lyophobic aqueous suspensions of pyrene-labeled nanoparticles (Py-NP) resulted in the formation of higher aggregates, as evidenced by DLS and ultramicroscopy. These appeared to remain aggregated due to hydrophobic interactions. This system could also be re-dispersed by sonication and re-aggregated in subsequent cycles of photo-excitation and sonication.

Maßgeschneiderte polymerisierbare Tenside zur simultanen Stabilisierung und Oberflächenfunktionalisierung von Nanopartikeln in der Miniemulsionspolymerisation

Im Rahmen dieser Arbeit wurden verschiedene funktionale, polymerisierbare Tenside (Surfmere) synthetisiert, um unmittelbar und exklusiv die Partikeloberfläche in der Miniemulsionspolymerisation mit der gewünschten Funktion für weitere Anwendungen auszurüsten. Hierdurch ist es möglich, auf konventionelle Tenside, welche bedingt durch ihre Mobilität in einigen Anwendungen zu Schwierigkeiten führen, gänzlich zu verzichten. Zusätzlich bietet der Einsatz von Surfmeren eine höhere Kontrolle über die Lokalisation und Verteilung der Funktionalitäten auf der Partikeloberfläche, im Vergleich zum Einsatz von klassischen Comonomeren. rnThematische Schwerpunkte der Arbeit lagen in der Ausrüstung von Partikeloberflächen mit Haftgruppen (Phosphonsäuren) oder Fluoreszenzmarkern sowie der Aufbringung von Initiatorgruppen über Surfmere zur Synthese von Kern-Schale-Partikeln in einem zweistufigen Prozess. Bei allen neu synthetisierten Surfmeren wurde als polymerisierbare Einheit eine Methacrylamidgruppe gewählt, um Funktionalitätenverlust durch Hydrolyse auszuschließen.rnIm Bereich der Haftgruppen wurde gezeigt, dass der Einsatz von phosphonathaltigen Surfmeren die Kontrolle der Partikelgröße und Funktionalisierungsdichte in weiten Bereichen ermöglicht und langzeitstabile Dispersionen erhalten werden. Die Partikel wurden auf ihre Cytotoxizität und ihre biomimetische Mineralisierbarkeit hin untersucht.rnZum Nachweis der Copolymerisation des Surfmers mit dem Hauptmonomer wurde ein Phosphonsäure-Surfmer mit einem Farbstoff auf Naphthalimidbasis synthetisiert. Dies ermöglichte den Nachweis der Copolymerisation mittels Gelpermeationschromatographie.rnZur Fluoreszenzmarkierung von Partikeloberfläche wurden erstmals Surfmere realisiert, die in der Kopfgruppe eine BODIPY-Einheit, welche in 2 oder 2,6-Position sulfoniert wurde, als Fluorophor tragen. Der Polymerisationsbeweis wurde durch HPLC-Messungen geführt und die Lokalisation auf der Partikeloberfläche durch Quenchungsexperimente verifiziert. rnDes Weiteren wurde ein kationisches Surfmer synthetisiert, welches nahe der Kopfgruppe eine Bromo iso-buttersäureeinheit zur AGET-ATRP-Initiierung trägt und somit potentiell zum Aufbau von Kern-Schale-Morphologien befähigt ist.